Autoimmune thyroid diseases (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT) are currently treated only symptomatically and not by targeting the mechanisms causing disease. Our long-term goal is to design precision-targeted therapies for AITD by blocking presentation of thyroidal antigens to T-cells. In order to block antigen presentation we are targeting HLA-DRb1-Arg74, which we have previously shown to be the key HLA-DR pocket that presents thyroidal peptides triggering AITD. During the last grant period we made significant progress towards our long-term goals: (1) We identified the key TSHR peptide epitope causing GD; (2) We identified Cepharanthine as a compound that blocks DRb1-Arg74 and prevents AITD in a mouse model; (3) We identified D-peptides that block peptide binding to DRb1-Arg74; (4) Using genome-wide approaches we identified new AITD susceptibility genes (e.g. ARID5B); (5) We identified genetic-epigenetic interactions triggering AITD. Building on the progress made in the previous grant period we propose to develop new therapeutic approaches for AITD by blocking antigen presentation. Our hypothesis is that presentation of pathogenic Tg/TSHR peptides to T-cells within the DRb1-Arg74 pocket we discovered is key to triggering AITD, & that blocking peptide binding to this pocket can be used to treat/prevent AITD. Our specific aims are: Specific Aim 1: We will enhance the potency and efficacy of Cepharanthine in blocking antigen presentation within DRb1-Arg74 by creating modified Cepharanthine analogs (MCA's). We will use in silico methods to design MCA's; MCA's will be confirmed using our uniquely designed ELISA and cell based assays, and using ex vivo and in vivo experiments in ?humanized? DR3 mice in which we will induce autoimmune thyroiditis. Specific Aim 2: We will generate monoclonal antibodies (MAb's) targeting the HLA-DRb1-Arg74 ? Tg.2098 complex. This approach is based on our findings that presentation of the Tg peptide Tg.2098 by DRb1-Arg74 is the key step in triggering AITD. The MAb's we produce will be screened by our in vitro ELISA and cell based assays, and confirmed ex vivo and in vivo in our DR3 ?humanized? mouse model of AITD. Specific Aim 3: We will validate that MCA's & MAb's block antigen presentation in patients with AITD. We will validate MCA's and MAb's by testing their ability to inhibit T cell recall responses to thyroidal antigens in peripheral blood mononuclear cells (PBMC's) isolated from AITD patients that are positive for DRb1-Arg74. In summary, our multidisciplinary translational project builds on the knowledge gained in the previous grant period. Our goal is to pursue preclinical development of novel therapies for AITD based on blocking antigen presentation by HLA-DRb1-Arg74. Our collaborative team has the capacity, experience, & expertise to achieve the aims of our proposal. The main advantage of our therapeutic approach is that it is both selective since only T-cells recognizing pathogenic thyroidal peptides are targeted and personalized since only patients carrying the HLA-DRb1-Arg74 will be treated. Our translational studies will hopefully lead to novel therapies for AITD.